Concordance of experimentally mapped or predicted Z-DNA sites with positions of selected alternating purine-pyrimidine tracts.

The recent electronmicroscopic and biochemical mapping of Z-DNA sites in phi X174, SV40, pBR322 and PM2 DNAs has been used to determine two sets of criteria for identification of potential Z-DNA sequences in natural DNA genomes. The prediction of potential Z-DNA tracts and corresponding statistical analysis of their occurrence have been made on a sample of 14 DNA genomes. Alternating purine and pyrimidine tracts longer than 5 base pairs in length and their clusters (quasi alternating fragments) in the 14 genomes studied are under-represented compared to the expectation from corresponding random sequences. The fragments [d(G X C)]n and [d(C X G)]n (n greater than or equal to 3) in general do not occur in circular DNA genomes and are under-represented in the linear DNAs of phages lambda and T7, whereas in linear genomes of adenoviruses they are strongly over-represented. With minor exceptions, potential Z-DNA sites are also under-represented compared to random sequences. In the 14 genomes studied, predicted Z-DNA tracts occur in non-coding as well as in protein coding regions. The predicted Z-DNA sites in phi X174, SV40, pBR322 and PM2 correspond well with those mapped experimentally. A complete listing together with a compact graphical representation of alternating purine-pyrimidine fragments and their Z-forming potential are presented.     

Sodium-alanine cotransport in oocytes ofXenopus laevis: Correlation of alanine and sodium fluxes with potential and current changes

Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [¹⁴C]-l-alanine,²²Na⁺ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition.     

Osmotic water permeability through liposomes in the presence of α1-acid glycoprotein

¹-acid glycoprotein (orosomucoid) from human blood serum was isolated in pure form and then reconstituted into large multilamellar liposomes, consisting of a binary mixture of hen-egg phosphatidylcholine and cholesterol. These liposomes were found to be osmotically sensitive. The osmotic water permeability of proteoliposomes was determined by light-scattering measurements of the osmotic volume changes after mixing with hyperosmotic solutions of potassium salts and aminoglycoside antibiotics. The initial rate of water outflow was measured as a function of glycoprotein concentration in the mixture for the preparation of proteoliposomes. This can serve as an indication for membrane permeability to the solutes used in these experiments. It was shown that aminoglycoside antibiotics passed much faster across the membrane than potassium salts, in the presence of glycoprotein in the liposomes. A recognition pattern in the osmotic behavior of these proteoliposomes was assumed.     

Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper

A solid-phase method for simultaneous sequencing of ten or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173-1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO4 (T greater than Pu) and NH2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation.     

Synthesis of the mitogenic S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylpentapeptide from Escherichia coli lipoprotein

The N-terminal pentapeptide of the lipoprotein from the outer membrane of Escherichia coli was obtained by coupling S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine to O-tert-butylseryl-O-tert-butyl-seryl-asparaginyl-alanine tert-butyl ester followed by deprotection with trifluoroacetic acid. The tetrapeptide was built up from alanine tert-butyl ester with N-9-fluorenylmethyloxycarbonyl protected amino acids. S-[2,3-Bis(palmitoyloxy)propyl]-N-palmitoylcysteine was obtained from N,N'-dipalmitoylcystine di-tert-butyl ester via reduction to the thiol, and S-alkylation with racemic 3-bromo-1,2-propanediol followed by esterification with palmitic acid in the presence of dicyclohexylcarbodiimide/dimethylaminopyridine and deprotection with trifluoroacetic acid. The compounds were characterized unequivocally by 13C-NMR and mass spectra. The diastereomers of S-[2,3-bis(palmitoyloxy)propyl]-N-palmitoylcysteine tert-butyl ester with opposite configuration at the propyl-C-2 atom could be separated on a silica-gel column.     

Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.     

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed.Besides the 8 S cytosol estrogen $\text{receptor}$, there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α₁-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the $\text{receptor}$ increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α₁-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components.During the course of these experiments, it has been observed that the increase of the estradiol $\text{receptor}$ is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.     

The Avena geo-curvature test

Summary The Avena geo-curvature test is a bioassay for auxin-type growth regulators. Etiolated oat coleoptiles are used and the test is conducted in special perspex trays under diffuse daylight. The coleoptiles, with the primary leaves intact, are arranged in grooves on the trays in 4 series of 6 coleoptiles each, and cut to a size of 10 mm. The test substance is applied on an agar strip covering only the lower halves of the apical cut surfaces of each series. After a 4-hour stay in the dark in moist Petri dishes the coleoptile cylinders are shadowgraphed on a 35-mm photographic film. The curvatures are measured from an enlarged projection (10 times natural size) of the shadowgraphs.The lowest IAA concentration which can be determined is around 30 to 60 g/l, i.e. about 1 to 2 ng IAA. The concentration response curves follow a logarithmic course up to 1,000 g/l. The standard error of the mean in a test series comprising 6 coleoptiles is on an average ± 7%. The simple and quick procedures make it possible for a worker to test 60 to 80 fractions a day.Dedicated to Professor Hans Söding on the occasion of his seventieth birthday. The senior author expresses his special gratitude for having been initiated by Professor Söding in the study of auxins.present address: Dpt. of Biology Princeton Univ. Princeton, N. J. 08540, U.S.A.